Bioanalysis for Biocatalysis: Multiplexed Capillary Electrophoresis鈥揗ass Spectrometry Assay for Aminotransferase Substrate Discovery and Specificity Profiling

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• 作者：Gleb G. Mironov ; Antony D. St-Jacques ; Alexander Mungham ; Matthew G. Eason ; Roberto A. Chica ; Maxim V. Berezovski
• 刊名：Journal of the American Chemical Society
• 出版年：2013
• 出版时间：September 18, 2013
• 年：2013
• 卷：135
• 期：37
• 页码：13728-13736
• 全文大小：451K
• 年卷期：v.135,no.37(September 18, 2013)
• ISSN：1520-5126

文摘

In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections鈥搃ncubation鈥搒eparation鈥揺nzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion. We used the MINISEP-MS assay to profile the substrate specificity of three aminotransferases (E. coli aspartate aminotransferase, E. coli branched-chain amino acid aminotransferase, and Bacillus sp. YM-1 d-amino acid aminotransferase) for 33 potential amino acid substrates and to measure steady-state kinetics. Using MINISEP-MS, we were able to recapitulate the known substrate specificities and to discover new amino acid substrates for these industrially relevant enzymes. Additionally, we were able to measure the apparent K_M and k_cat parameters for amino acid donor substrates of these aminotransferases. Because of its many advantages, the MINISEP-MS assay has the potential of becoming a useful tool for researchers aiming to identify or create novel enzymes for specific biocatalytic applications.